The structural connectivity mapping of the intralaminar thalamic nuclei

The intralaminar nuclei of the thalamus play a pivotal role in awareness, conscious experience, arousal, sleep, vigilance, as well as in cognitive, sensory, and sexual processing. Nonetheless, in humans, little is known about the direct involvement of these nuclei in such multifaceted functions and their structural connections in the brain. Thus, examining the versatility of structural connectivity of the intralaminar nuclei with the rest of the brain seems reasonable. Herein, we attempt to show the direct structural connectivity of the intralaminar nuclei to diencephalic, mesencephalic, and cortical areas using probabilistic tracking of the diffusion data from the human connectome project. The intralaminar nuclei fiber distributions span a wide range of subcortical and cortical areas. Moreover, the central medial and parafascicular nucleus reveal similar connectivity to the temporal, visual, and frontal cortices with only slight variability. The central lateral nucleus displays a refined projection to the superior colliculus and fornix. The centromedian nucleus seems to be an essential component of the subcortical somatosensory system, as it mainly displays connectivity via the medial and superior cerebellar peduncle to the brainstem and the cerebellar lobules. The subparafascicular nucleus projects to the somatosensory processing areas. It is interesting to note that all intralaminar nuclei have connections to the brainstem. In brief, the structural connectivity of the intralaminar nuclei aligns with the structural core of various functional demands for arousal, emotion, cognition, sensory, vision, and motor processing. This study sheds light on our understanding of the structural connectivity of the intralaminar nuclei with cortical and subcortical structures, which is of great interest to a broader audience in clinical and neuroscience research.

Prefrontal thalamic projection site (from oxford thalamic atlas) PMTPs Premotor thalamic projection site (from oxford thalamic atlas) PPTPs Posterior parietal thalamic projection site (from oxford thalamic atlas) OTPs Occipital thalamic projection site (from oxford thalamic atlas) TTPs Temporal thalamic projection site (from oxford thalamic atlas) PRMTPs Premotor thalamic projection site (from oxford thalamic atlas) PD The thalamus is a mysterious and fascinating structure in the brain. It is intricately connected to various regions of the brain through projection fibers. The thalamus can be divided into four major groups of nuclei-the anterior, medial, lateral, and posterior groups-each with its specific function [1][2][3][4][5] (Fig. 1, Supplementary Table 1). But within the medial group, there is a subgroup that stands out, the intralaminar nuclei (ILN). These nuclei are located within a unique and remarkable fiber pathway called the internal medullary lamina and are known to have a global influence on mental and cognitive function (Fig. 2). They diffusely project to different brain areas [6][7][8][9] , which enables them to control the transmission of information and the synchrony of the cortex 10 . The ILN also acts as a bridge between the brainstem and the cortex, facilitating rapid communication and processing of various functions such as awareness, conscious experience, perception, arousal, vigilance, sleep, visual, sensorimotor, attention, and sexual processing [11][12][13][14][15][16][17][18][19][20] . They are not only intriguing but also crucial in the understanding of disorders of consciousness, psychiatric conditions, and neurodegenerative diseases. They have even been identified as a target for deep brain neuromodulation in treating disorders of consciousness, highlighting their importance in brain functioning 21 .
Despite the ILN's crucial role in multiple brain functions, a detailed understanding of each individual ILN and its structural connectivity with other brain regions remains elusive. While the ILN structural connectivity maps have been extensively determined in animal research 19 , they remain a mystery in humans. A handful of human studies have investigated the structural connectivity of the ILN as a group or complex 22,23 , but none have delved into the specific connectivity patterns of individual nuclei. This lack of information leaves many questions unanswered, and a deeper understanding of the structural connectivity of each ILN is essential to grasp their specific functions fully. Understanding structural connectivity can provide valuable insights, but we can only truly unravel the intricacies of the ILN's role in the brain by studying the individual nuclei. Therefore, it is essential to determine the structural connectivity patterns of the intralaminar nuclei in humans to understand further its role in various brain functions and the development as well as treatment of neurological and psychiatric disorders.
In the present study, we set out to unravel the mysterious ILN by delving into its structural connectivity patterns. We constructed detailed fiber connectivity maps for the five ILN nuclei using data from 730 healthy volunteers from the human connectome project (HCP) 24 . The study hypothesizes that the ILN communicates with various subcortical and cortical areas and constitutes different networks to facilitate diverse behavioral demands. www.nature.com/scientificreports/ T1 to MNI space was computed in the registration procedure. In the next step, the non-linear registration was performed. In the third step, the inverse of the MNI to the non-diffusion space was calculated to register the nuclei into the native subject space 29,30 . The nuclei transformation allowed further diffusion calculations into the subject native space while maintaining high data quality and reducing registration interpolation errors 31 .

Methods
Diffusion-fit. The preprocessing included distortion and motion correction within the HCP pipeline [32][33][34] . The diffusion fit was performed using the FSL DTIFIT. The diffusion fit yielded color-coded FA maps in each subject. The visual inspection of each subject's FA map determined the quality of the diffusion fit.  www.nature.com/scientificreports/ Diffusion reconstruction. The reconstruction used a multishell model (three fibers per voxel, rician noise) 35 . The default noise was rician noise. Each subject's diffusion reconstruction was parallelized using sun-gridengine (fsl_sub). The whole-brain multishell reconstruction required similar parameters for each subject.
Probabilistic tractography. The probabilistic tractography was applied using FSL-probtrackx 36 . The probability diffusion algorithm repetitively samples from the distributions of voxel-wise principal diffusion directions by computing each time a streamline through the local samples to generate a probabilistic streamline or a sample from the distribution on the location of the true streamline. FMRIB's Diffusion Toolbox (FDT) builds up the histogram of the posterior distribution on the streamline location or the connectivity distribution 36 . The probtrackx parameters included curvature threshold 80° (0.2), sample number 5000, step length 0.5, and a maximum number of steps: 2000. In the direct diffusion tractography, all streamlines passing through other nuclei were excluded from depicting only direct connections to the rest of the brain. The resulting tractograms were normalized by dividing them by the waytotal and multiplying them by 100.
Native-subject-space Tractogram registration to MNI Space. The registration of the native-subject-spaced tracts to the MNI space relies on a combination of linear and non-linear registration steps (Supplementary Text). For each subject, a non-diffusion map to the structural T1 and T1 to 1 mm MNI brain was registered using the flirt linear registration method implemented in the FSL. The non-linear registration uses the output parameters from the linear registration and performs finer alignment to the MNI space using the fsl-fnirt method implemented in the FSL. Furthermore, using the above-generated non-linear and linear registrations, the fsl-applywarp tool was used to register the native-subject-spaced tractography maps to the MNI brain.
Group fixed effect analysis. The non-diffusion volume of each subject was coregistered to MNI brain space using a combination of linear and non-linear transformations described above. The resulting transformation matrices were then applied to the native-tractograms to align to the MNI space. The aligned tractograms from all subjects depicted the group fixed effect maps.
Visualization. The resulting group fixed effect maps were visualized using the mricron package. The group fixed effect maps were minimally thresholded (thr 1) to remove weaker and spurious probabilities. The brain's axial, sagittal, and coronal views visualize each fixed-effect-map (Figs. 3, 4, 5, 6, 7, 8, Supplementary Figs. 1-6). The labeled 2D slices were shown in axial, sagittal, and coronal views. The connectivity on the cortical surface visualizes the endpoints of the touching tract volume mesh. The 3D tracts (rendering and surface) illustrations are arranged side to side in six different viewpoints, i.e., left, right, posterior, anterior, inferior, and superior. The rendering views were visualized using the surfice software package.
Anatomical atlas label search and assignments. The anatomical assignments of the fixed effect maps (thr 1) determined specific labels for cortical, subcortical, white-matter tracts, and cerebellar projections. The anatomical labeling procedure employed the Harvard-Oxford cortical-subcortical structural atlas [38][39][40][41] , the JHU white matter tractography atlas 42 , the Jülich histological atlas [43][44][45] , oxford thalamus atlas, subthalamic nucleus atlas, oxford manova striatal structural atlas, Human sensorimotor tracts label atlas, XTRACT HCP probabilistic tract atlas, and the cerebellar atlas in MNI152 space after normalization with FNIRT 46 available within FSL 47 . The oxford thalamus atlas assigns the connectivity localization within the thalamus 48 . The detailed assignments of the brainstem nuclei relied on the brainstem navigator atlas 49 . In the results, the description of pathways and connections uses the words ('project/projected') to describe diffusion data-driven structural connectivity, which doesn't distinguish between the incoming and outgoing connections to the individual ILN due to the underlying methodological limitations.

Results
Intralaminar nuclei of the thalamus. The ILN enveloping the medially located mediodorsal nucleus (MD) refers to assembling nuclear structures within the thalamus' internal medullary lamina (IML) (Fig. 2). The IML is a remarkably constructed myelinated fiber pathway in the center of the thalamus. It appears as a Y-shaped white stripe in axial sections and delimits the different thalamic territories that form the medial, lateral, and anterior groups of thalamic nuclei. In general, the ILN has been associated with the truncothalamic complex, as they constitute a major part of the so-called 'nonspecific' thalamocortical system that relays the activity of the brainstem reticular formation to widespread cerebral cortical areas. Depending on the referring anatomist 50-52 , the ILN can be divided into two or three groups (Fig. 2). The first is the central medial nucleus (CeM), located at the midline between the ILM and the mediodorsal nucleus (MD). The second is situated laterally in the anterior part of the IML and includes the paracentral and central lateral (CL) nuclei. The third expands posteriorly in splitting the IML and includes the posterior intralaminar, centre median (CM), and parafascicular (Pf) nuclei. Some authors 2,51 further distinguish a small subparafascicular nucleus (sPf) within a splitting of the external medullary Iamina just ventral to the Pf proper.
Structural connectivity. Central lateral nucleus CL. The CL is the largest intralaminar nucleus in the morel atlas 28 , revealing the most confined connectivity of all ILN. The CL expands anteriorly to posterior, dorsally covering CeM, Pf, sPf, and CM (Fig. 2). The CL connectivity maps (Fig. 3, Supplementary Fig. 1 53,54 . In addition, CL possesses intrinsic thalamic connections to the prefrontal projection site (PTPs) and the temporal projection site in the Oxford thalamus atlas (TTPs). Interestingly, the inception of anterior thalamic radiation (ATR) also shows connectivity with CL. The Anterior and superior thalamic radiation show slight dominance in the left CL connectivity map in contrast to the right CL (Supplementary Table 10).
Centromedian/Centremedian nucleus CM. The CM is the second-largest nucleus in the ILN group. The CM is located in the central core, among other ILN, in sagittal view ventrally to CL, above Pf, sPf, and posterior to CeM (Fig. 2, Supplementary Fig. 1a,b). The CM projects to wider motor and sensory system brain areas, suggesting a key role in the motor system ( Fig. 4, Supplementary Fig. 2, Supplementary Table 3). The CM shows connectivity via the medial and superior cerebellar peduncle (MCP, SCP) with the brainstem (BS) and cerebellar lobules, i.e., crus I, crus II, V, IX, I-IV, VIIb. The lobule I-IV and V are part of the somatotopic motor system 55 . However, con- The CL projections consist of SC, mesencephalic reticular formation (Dorsal part), and fornix. PTPs prefrontal thalamic projection site in oxford thalamus atlas, TTPs temporal thalamic projection in oxford thalamus atlas, ATR anterior thalamic radiation, BS brainstem, SC superior colliculus (Superior dorsal portion of the tectum in the midbrain). The detailed anatomical assignments are given in Supplementary www.nature.com/scientificreports/ nections to the other cerebellar lobules suggest a broader functional integration with working memory (crus-I/ II) and multisensory integration (VIIb). Furthermore, CM connects to the corticospinal tract (CST); however, the CST does not reach the cortex. Interestingly, the CM connectivity to the amygdala superficial group (Ag) is found. We have noticed similar connections to the pallidum in line with these results. The brain stem nuclei, i.e., raphe nucleus, periaqueductal gray, cuneiform nucleus, inferior colliculus, Inferior medullary reticular formation, inferior olivary nucleus, parabrachial nucleus, prabigeminal nucleus, mesencephalic reticular formation, pedunculopontine nucleus, sustantia nigra, vestibular nuclei, viscero-sensory-motor nuclei, and ventral tegmental area show prominent connectivity with CM (Supplementary Table 7). The cerebellar lobule Left Crus II shows a slightly higher overlap with Left CM, in contrast with Right Crus II (Supplementary Table 8).
Central medial nucleus CeM. The CeM is the third-largest nucleus within the ILN group. In the dorsal view, CeM is located below CL, next to the medial wall of the brain hemisphere (Fig. 2). In the sagittal view, the CeM locates itself at the anterior border and neighboring the posteriorly situated Pf (Fig. 2). The CeM projects (Fig. 5, Supplementary Fig. 3, Supplementary Table 4) to the anterior commissure (AC) and then further via the ATR to the orbito-frontal cortices, especially in the Brodmann areas (BA) 11. The tracts migrate from the AC to the medial temporal lobes, encircle the amygdala (Ag), and connect to the hippocampus gyrus. The CeM further projects subcortically to the pallidum, putamen, and caudate, as well as to the fornix and cingulum. Posteriorly,   Table 7).
The Superior parietal lobule 7P shows slight dominance in the right CeM connectivity map compared to the left CeM (Supplementary Table 11).
Parafascicular nucleus Pf. The Pf, the second largest nucleus like CM, lies adjacent to the CeM at the posterior side (Fig. 2) and is sagittally located below the CL and neighboring CM. The Pf projections consist of SCP, ICP, IOFF, aIOFFf, pIOFFf, MCP, CPCF, CSF, ILF, BS, HEC, UF, SPL-5M, CB, PTPs, SS, MGB, fornix, HS, Ag, OR, and the FC (Fig. 6, Supplementary Fig. 4, Supplementary Table 5). The Pf projections are, moreover, similar  Similar connections of the adjacent CeM and Pf suggest that they share identical thalamus peduncles/ radiations and project to similar brain areas due to their spatial proximity. Interestingly, both share connections to important brain areas, including visual, temporal, and frontal cortices. The latter are among other brain regions that contain significant nodes in the human default mode brain network. The CeM and Pf connectivity similarity possibly provides connectivity demands for the highly activated default mode network facilitating arousal, awareness, and other functions. The Visual cortex V1 BA17 shows slight dominance in the right Pf connectivity map compared to the left Pf (Supplementary Table 11). The brain stem nuclei, i.e., raphe nucleus, parabrachial nucleus, prabigeminal nucleus, mesencephalic reticular formation, pedunculopontine nucleus, vestibular nuclei, and viscero-sensory-motor nuclei show prominent connectivity with Pf (Supplementary Table 7). Subparafascicular nucleus sPf. The sPf is the smallest nucleus and lies as a tiny elliptical-shaped extended space under the Pf (Fig. 2). However, the sPf displays some unique connectivity patterns compared to other nuclei   Table 6). The subcortical projections include caudate, putamen, and pallidum. These connections include the BS and corticospinal tract (CST). The cortical projections via the CST and superior longitudinal fasciculus (SLF) enter cortical areas, including the secondary somatosensory cortex (SSC), the primary somatosensory cortex (PSC), the primary motor cortex (PMC), insular cortex (IC), the precentral gyrus (PrG) and postcentral gyrus (PoG), the superior parietal 7PR and the inferior parietal lobule. Interestingly, most of these brain areas are also part of the broader somatosensory system, permitting motor and sensory computation as well as spatial orientation 56 and awareness of the somatotopic events 57 . The superior parietal lobule cortical areas show slight dominance in the right sPf connectivity map compared to the left sPf (Supplementary Table 11). The brain stem nuclei, i.e., Inferior olivary nucleus, and prabigeminal nucleus, pedunculopontine nucleus, show prominent connectivity with Pf (Supplementary Table 7).
ILN specific connectivity. The ILN connectivity maps showed partly overlapping but specific projection patterns (Fig. 8, Supplementary Figs. 6-7, Supplementary Tables 2-11). Overall, the connectivity map reveals that all ILN connect to the brainstem, where all sensory afferents enter the brain. The CeM and Pf displayed similar connectivity patterns to the brainstem, cerebellum, visual, and frontal cortices. The CM directly projects mainly to the brainstem and cerebellum. The CL remains strictly confined to connectivity with the SC in brainstem. The www.nature.com/scientificreports/ sPF specifically contains motor pathway projections from the brainstem, cerebellum to the motor cortices, possibly facilitating rapid motor planning, execution, and action.

Discussion
The results gathered in this work reveal specific and partly overlapping connectivity patterns spanning a wide range of subcortical and cortical areas by utilizing high-resolution diffusion data in an HCP sample of 730 healthy subjects to determine the nuclei-specific connectivity of five ILN. The central medial nucleus (CeM) and the parafascicular nucleus (Pf) have particularly broad connectivity to the brainstem, cerebellum, subcortex, visual and frontal cortices, while the centromedian (CM) connects mainly to the subcortical motor system, including the brainstem and the cerebellum. The central lateral (CL) connects to the superior colliculus and fornix. The subparafascicular nucleus (sPF) presents specific projections to the basal ganglia, motor, somatosensory, parietal, and insular cortices. In short, the ILN offers overlapping and diverse connectivity patterns, suggesting variations in their functional involvement. The results of this research paint a picture of nuclei-specific ILN connections to subcortical and cortical areas, providing a deeper understanding of the intricacies of the thalamus. In the discussion, the first section below compares the findings with the previous studies, followed by nucleispecific connectivity in animal tracer studies. The tracer connectivity description elevates the understanding of the diffusion-tractography-driven ILN connectivity maps. The third section discusses brain-wide connectivity maps and their functional associations as the ILN has been implicated in various brain functions, i.e., conscious state, arousal, visual, sensorimotor, and attention 13,19,58 . The last section discusses the study's limitations and challenges.
ILN connectivity. The ILN connectivity patterns demonstrate partly overlapping and nuclei-specific connections. The connectivity maps have shown that all ILN has prominent connections to the brainstem, highlighting the close relationship between the ILN and the brainstem, where all sensory information enters the brain. Remarkably, all ILN connects with the brainstem connections, which is important for numerous brain functions, including motor, sensory, arousal, and vigilance 16 . Despite being the largest ILN, CL shows a rather refined projection to the SC. While CM remains confined to the subcortical cerebellar and brainstem projections, the CeM and Pf connect to the frontal, visual, temporal, and subcortical brain regions, encompassing key areas of the default mode network nodes. The sPf outlines specific tracts to the somatosensory cortex encircling the sensorimotor network.

Comparison with previous diffusion and functional MRI studies.
This study highlights the nuclei-specific detailed connectivity (Fig. 8, Supplementary Tables 2-11), in contrast to previous work by Jang and colleagues and Lambert and colleagues, who combined all intralaminar nuclei to perform a structural connectivity mapping of the ILN 22,23 . These studies 22,23 fails to distinguish between the different nuclei of ILN. For instance, Jang et al. and colleagues used the Oxford thalamic atlas to delineate a single ILN mask containing all ILN. Using a single ILN mask that encircles all regions of ILN cannot be directly compared with our nuclei-specific connectivity maps. However, our nuclei-specific combined connectivity maps (Fig. 8, Supplementary Figs. 6, 7) reveal similarities as well as some distinct differences in contrast to combined ILN connectivity maps reported by Jang et al. and colleagues 22 . In particular, we found new connections to specific visual cortices, i.e., V1-V2, parts of the brainstem, and cerebellum lobules (I-IV, V, VIIb, Crus I, and Crus II). A significant difference also exists regarding the data quality of the HCP, the number of subjects, and the state-of-the-art analysis methodology, i.e., diffusion spectrum imaging/multishell reconstruction 59 .
Lambert and colleagues 23 used euclidean distance to characterize probabilistic tractography distributions derived from diffusion MRI of 40 subjects from the HCP. Their study generated 12 feature maps to delineate individual thalamic nuclei, extracted tractography profiles for each and calculated the voxel-wise tractography gradients. Such feature maps do not delineate nuclei-specific maps of the intralaminar group. However, the combined midline-intralaminar feature map was found to have connections to the orbitofrontal cortex, entorhinal and calcarine cortices, as well as to the striatum, amygdala, and ventral mesencephalon 23 . Basile and colleagues 60 performed in-vivo super-resolution track-density imaging using 210 subjects from the human connectome project. Study 60 examined the structural and functional connectivity of combined masks of CM/Pf and MD/CL; therefore, it is not directly comparable to individual intralaminar connectivity patterns in our study 60 . However, combining the structural and functional connectivity of the CM/Pf complex to the middle and superior frontal gyri, supplementary motor, sensory regions, middle cingulate cortex, and insula aligns with our Pf connectivity (Fig. 6). Notably, the previous studies 22,23,60 offer a basis for comparison and reliability of this study's observed connectivity patterns of the ILN group.
Alignment with animal studies. In animals, an anterograde tracer injection displays their terminal's detailed nuclei connectivity patterns and passing fibers communicating to other brain areas. Numerous tracer studies existed on mouse thalamic tracking 61 , macaque thalamic connectivity, and several animal anterogrades [62][63][64][65][66][67][68] . Using such a robust tracer technique, the CeM shows projections to the rat's brainstem 69,70,[70][71][72] . CeM also shows projections to the amygdala, putamen, caudate, and cerebellum 19 . In the cortical tracer studies 19 , the CeM projects cingulate cortices in rats, cats, and monkeys to the perirhinal cortex, the entorhinal cortex, the visual areas, and the claustrum. Also, the CeM shows widespread projections in the rats across the different cortical areas 73 . The CL projects to the rat's brainstem. Indirect projections via transthalamic fibers to the prefrontal and temporal cortices align with the reported animal work 19 . Remarkably, our results show CL projections to the SC in brainstem, as reported in ILN-SC studies 53 www.nature.com/scientificreports/ the connectivity described in animal studies. However, a detailed point-by-point comparison is unattainable, as the tracer data cannot be normalized in human space and only has limited access for comparison.
The brain-wide connectivity and functional associations. The centrally located ILN establishes interconnectivity within the thalamus, enabling highly privileged access to various cortical areas 2,74,75 . The integration and synchronization of multiple brain areas [76][77][78] can result in a stream of consciousness 79 . All ILN projections align with the literature, suggesting their fundamental role in conscious processing and awareness. Thus, well-aligned with previous studies, the results depict ILN connectivity to the brainstem, basal ganglia, forebrain, and sensorimotor cortex 21,[80][81][82][83][84][85] . The results revealed that the ILN cumulatively connects via other thalamic nuclei and subcortical pathways to a wide range of cortical areas, which align with the system-wide arousal circuitry 58 . The arousal circuitry encircles widespread connections, including the brainstem, thalamus, hypothalamus, basal forebrain, and cerebral cortex 58,86 . It is widely accepted that arousal is required to process visual attention 87 . The ILN resides next to the most prominent thalamic nuclei, i.e., the mediodorsal nucleus (MD), which primarily connects with the prefrontal cortices, and these activations result in the wakening of the animal. Our results also found that the sPf nuclei project to motor, sensory, and parietal cortices. These findings agree with Jones's matrix-core theory of thalamic organization, in which the matrix nuclei (including ILN) serve as a binding locus with the cortex to achieve synchrony [13][14][15] and integration to perforce motor, sensory, parietal, frontal, and visual projections. 88,89 . Dystonia of the intralaminar midline complex causes fixed eye deviation, thought disorder, postural and autonomic disturbances 90 . The CM-Pf nuclei's underlying functions are mainly related to arousal, attention, and sensorimotor functions 91 . Attention involves wider brain areas, i.e., cerebellar lobules and the temporal lobe, to facilitate attention. In our study, CeM and midline nuclei project to the cingulate; they seem to play a role in effectively processing tactile-input/nociceptive information 92,93 .
The ILN connections and their underline implicated functions in the literature are discussed in more detail below.
The brainstem is the most engaged projection site. All ILN reveal connectivity with the brainstem (Fig. 8 Table 7). This finding aligns well with the previous research work. The previous research work shows that the ILN receives extensive inputs from the brainstem 19,81,[94][95][96] , as the ILN constitutes the dorsal pathway of the ascending reticular activating system of the brainstem to the cortex 83,97 . It is known from animal work that the brain alerts while performing an electric stimulation on the midbrain reticular formation and intralaminar nuclei. In humans, ILN shows activations during rest in an attention-demanding task implicating that the ILN and brainstem are important in arousal and vigilance 16 . The brainstem reticular formation covers most arousal-specific nuclei 58 , including locus coeruleus, raphe nuclei, and ascending arousal brainstem nodes 98,99 . The ILN receives inputs from most arousal-specific nuclei 58 , including locus coeruleus, raphe nuclei, and ascending arousal brainstem nodes 98,99 , and connects them with different cortical areas 100 .
Connectivity to sensorimotor cortices. Several studies show the CM and sPf connections with the basal ganglia, motor, and sensory cortices 18,19,22,[69][70][71][72]74,75,101 . Similarly, we found CM and sPf projections in the subcortical and cortical sensorimotor networks. The electrical stimulation of ILN induces head motion, eventually increasing responses to visual stimuli [102][103][104] . In a similar line of evidence, the Parent and Hazrati 105 work indicates that CM can effectively play an essential role in motor response modulation rather than sensory, visceral, emotional, or cognition-related functional processes. The motor modulation induces dopamine release from the striatum 106 , which seems reasonable for the CM-pallidum projections. Degeneration of caudal ILN nuclei results in progressive supranuclear palsy and Parkinson's disease 107 . The determined CM and sPf somatosensory connections align well with Henderson's study and play a part in motor control 94 and associative-limbic motor functions 19 .
Connectivity to SC. According to Jones's matrix core theory, the CL nucleus is a matrix nucleus, and attention employs such nuclei for higher-order computation [13][14][15] . While the CL reveals connectivity with the SC. In coordination with the thalamic reticular nucleus, pulvinar nuclei, and other brain areas, the SC might play a significant role in orienting, attentional focusing, attention selection, and attention implementation [108][109][110][111][112] . The SC continuously constructs discrete visual retinotopic fields and connects them with the pulvinar and lateral geniculate nucleus. Our analysis found connections of the CL with the PTPs and TTPs, suggesting a structural path between the SC communication with the parietal and temporal lobe. This supports the idea that the superior colliculus needs input from multiple brain areas to enable continuous visual field mapping. The CL to SC-thalamic projections are part of the arousal system, a converged forebrain circuit that controls orienting, defense (fight or flight behavior), and sensory-motor integration 54 . Visual awareness requires ILN involvement 113,114 as arousal directly correlates with pupil size, visual processing, on-off cortical dynamics 115,116 , and attention changes 87 . CL projects to the SC 54 to visual areas 53 , motor and arousal areas 117 to continuously shape the visual experience.
Connectivity to parietal cortices. Parietal cortices achieve sensorimotor integration by transforming visual maps into non-retinocentric coordinates through multisensory areas. For example, the multisensory parietal cortex transforms visual maps into non-retinocentric coordinates 118 . ILN connectivity with selected parietal cortices suggests their involvement with multisensory integration and the maintenance of multisensory integration and synchronization.
Connectivity to frontal cortices. The reticular formation connects to ILN, the basal forebrain, hypothalamus, and prefrontal cortices as areas are involved in arousal, control of attention, and sensorimotor function 19 www.nature.com/scientificreports/ The previous work demonstrates that the partial infarctions of the ILN cause cognitive deficits 19 , resulting in decreased flexibility in the employment of cognitive strategies, i.e., dysexecutive syndrome 119 .
CeM connectivity facilitates arousal and sleep. The thalamus acts as a hub for sleep for subcortical and cortical inputs 11 and contributes to slow sleep oscillations in humans 120 . The CeM and other brain areas also play a role in arousal studies 58 . Specific deep brain stimulation of midline/intralaminar nuclei interventions show heightened arousal, speech recovery, restored executive motor control, and improved feeding behavior after severe traumatic brain injury-induced minimally conscious state 21 . A recent study shows that the tonic and burst firing pattern of CeM neurons can modulate brain-wide cortical activity during sleep and provide dual control of sleep-wake states 11 . In the CeM, the connections with the frontal cortices, the brainstem, raphe nucleus, ventral striatum, VTA, and hypothalamus are neuronal substrates of sleep-wake states.
Neuromodulation and intralaminar nuclei. The central lateral (CL) nuclei show reduced consciousness in macaques after deep brain stimulation (DBS) 121 . The centromedian (CM) is a target nucleus for generalized or multifocal seizures for the neuromodulatory treatment using deep brain stimulation [122][123][124] . The neuromodulation of CM-Pf complex using deep brain stimulation for Tourette's syndrome is also an emerging target for treatment 125 . The DBS of Pf may modulate cognitive functions by inducing molecular-level gene expression changes in the prefrontal cortex 126 . The sPf stimulations show dopamine release modulation in the inferior colliculus of rats and are suggested to be involved in the auditory processing deficits associated with Parkinson's 127 .
Stimulation of the CM in people with epilepsy leads to activation of diffuse, cortico-cortical evoked potentials 128 .
The neuromodulation of the intralaminar nuclei using DBS may engage the nuclei-specific connected cortical and subcortical areas in various ways, leading to precise changes in behavior. For instance, the CM connects subcortically with almost all the basal ganglia nuclei, i.e., caudate, putamen, pallidum, substantia nigra, subthalamic nucleus, and cortically with sensorimotor, premotor 129,130 , and dorsolateral prefrontal cortex 131 . Therefore, a deeper understanding is warranted concerning the neuromodulation of each intralaminar nuclei and their combinations to further understand the impact on connected cortical brain regions. Such exploration may provide a more comprehensive understanding of the intralaminar nuclei's clinical implications, translational potential, and relevance in neurological and psychiatric conditions. It would also strengthen the validity of this study's observed structural connectivity of the intralaminar nuclei.

Limitations. Cortico-thalamo-cortical feedforward and feedback communication.
The complex corticothalamo-cortical feedforward and feedback interrelationships employ a layer-specific input and output mode 132 . However, due to methodological limitations, diffusion imaging cannot delineate such layer/column-specific details. The diffusion imaging captures coarser resolution and, therefore, cannot infer the layer/column-specific precise details like interlayer communicational architecture, axon collaterals, cortico-thalamic branching axons, extrathalamic axon branching to different cortical areas, and differentiation between the driver and modulator connections.
Erroneous estimations due to atlas-defined seed regions. Our results rely on histologically defined seed regions, which always include a bias. The parcellation of the thalamus is a challenging and unresolved question 133 . The atlas-based method depends on a limited number of post-mortem brains, therefore, cannot account for interindividual variabilities 134 . The atlas-defined regions can contain a mix of signals which may not be specific to the functional similarities. This mixing could become worse when multiple subjects are grouped after MNI normalization. Using a standard anatomical seed region does not account for internal nuclei architectonics that partially influences neighboring nuclei. These mixed signals are the main seed region for the diffusion and functional thalamo-cortical connectivity analysis and thus can lead to erroneous estimations 135,136 . In this study, the diffusion analysis uses the native subject space; only the fiber projections were transformed in the MNI space. The percentage volume of the left and right group fixed effect maps depicts a variable overlap (Supplementary Table 12). The CeM shows a high overlap with Pf, while CL shows only minimal overlap. We also noticed that the CM displays partly overlap with CeM, Pf, and sPf. Finally, a significantly higher overlap was observed for the Pf with CeM. In contrast, sPf shows a low overlap with other nuclei. All these different overlaps can be partly attributed to the bias of the atlas-defined seed regions, but cases of high probablity suggest reliable connectivity projections ( Supplementary Figs. 13).
A single nucleus may have specific sub-regions, and they can display variance in their projections to achieve a precise finely-grained functional influence in the brain 64 . The nuclei sub-regions can only contain a few thousand neurons due to the smaller size of the nuclei. Therefore, their subregional projections and anatomical-specific localization are lacking due to resolution limits in MRI.
Structural connectivity and different tracking algorithms:. Different algorithms can be applied to infer structural connectivity from diffusion MRI data, such as probabilistic tracking, deterministic tracking, and unscented Kalman filter (UKF) tracking algorithms. These algorithms can potentially affect the experimental results when evaluating structural connectivity. In the study, we only used a probabilistic tracking algorithm considering the vast amount of data, which requires computational resources and extended disk space. However, it will be imperative and informative to validate the results using multiple methods to ensure the robustness of the findings in a future study. Additionally, it is important to consider the specific features of each algorithm when interpreting the results and to be aware of each method's potential limitations and sources of bias. www.nature.com/scientificreports/ Structural connectivity and diffusion MRI of the brain. Due to a limited resolution of 1.25 mm isotropic, we capture the structural connectivity maps in a young, large healthy cohort. In addition, the MR coils usually yield a higher signal-to-noise ratio on the cortical ribbon than on the subcortical structures. However, as the spatial resolution and sequence optimization for subcortical structures improve, we may infer more specific fiber paths and their configurations 137 . For example, at the moment, we have no insights on the specific tangential connections up to the level of the gray matter, i.e., U-fibers. Furthermore, we do not speculate how the current diffusion tractography results correlate with the myloarchitecture of the brain. There also remains a poor understanding of whether the projections originate from the specific nuclei or come from other brain areas. We cannot observe such connectivity linkage due to limited resolution. Hopefully, future work in the field will provide high-resolution data and better methods to precisely delineate the described connectivity results. The diffusion tractography can give an erroneous estimation of connections by having false positives or negatives 36,138 . The false positives can be partly corrected using methods like thresholding, visual inspection, comparison with previous literature, cross-validation, use of multiple algorithms and parameters, and statistical correction. The described connectivity maps use thresholding, visual inspection, and comparison with described connectivity patterns in literature. The statistical correction (FWE p < 0.05 and p < 0.001) (Supplementary Figs. [8][9][10][11][12]Supplement 139 . This percentage may vary since humans have very different cortical architecture. Therefore, the described connectivity should be looked up with the methodological awareness of diffusion spectrum imaging and under a constrained measurement parameter setting, which can induce drastic differences in the results.
The study provides a comprehensive understanding of the structural connectivity of the intralaminar nuclei, but the reproducibility of these findings in clinical populations remains to be determined. More research is needed to confirm that these connections can be consistently observed in clinical data. However, we can increase the chances of reproducing these findings in clinical populations by using similar methods and data acquisition techniques to the HCP study.
In summary, the structural mapping in the brain anticipates better and more precise delineation of connectivity using high-resolution data, high-field MR advancement, better diffusion reconstruction, tractography, and empirically referenced in-vivo findings of the population-level histological work. Therefore, our findings and fiber distributions underlining the anatomical-wiring information remain tentative.

Conclusion
The ILN structural connectivity suggests a critical nuclei group in the structural path from the brainstem and the cerebellum to specific cortical areas. They display overlapping and nuclei-specific connectivities to specific cortical-subcortical cerebellar and brainstem sites. The sPf connectivity appears as a key in the somatosensory processing unit, covering the brainstem, cerebellar areas, basal ganglia, and specific cortices. Interestingly, CM seems to be an essential component of the subcortical somatosensory system. The CM connections are similar to the sPf projections but remain confined to the subcortex. The CeM and Pf show similar connectivity projections with a slight variance. The CeM projections, compared to the Pf, show dense intrathalamic connectivities. The Pf displays slightly more spacious cortical connectivities in comparison with the CeM. However, both project to the visual, frontal, and temporal cortices and give access to some default mode network nodes. The CeM and Pf notably project through the subcortical system to ILF, UF, and IOFF, allowing broad access to brain areas and enabling visual and cognitive processing. It is worth noting that the CL shows a precise projection to the SC in the brainstem. The five ILN diffusion-defined connectivity maps span a wide range of subcortical and cortical areas. The findings align with the known structural core for various functional demands like arousal, emotion, cognition, sensory, vision, and motor processing. The described ILN connectivity relies on diffusion-driven analysis and does not directly describe the axonal path. However, knowing the anatomical connections in this study may facilitate the investigation of the potential role of ILN in healthy and disordered brains.